Interleukin-15 (IL-15) was identified as a new cytokine able to replace IL-2 in supporting the proliferation of a murine T cell line (1,2). Both cytokines belong to the four alpha helix bundle family (3). IL-15 was initially found to mimic most of the in vitro activities elicited by IL-2 in vitro, including induction of proliferation and cytotoxicity by activated T cells (1) and NK cells (2,4), co-stimulation of B cell proliferation and immunoglobulin synthesis (5) and chemo-attraction for T cells (6). This redundancy is explained by the common usage within their functional receptors of the IL-2Rβ/γ signaling complex. This IL-2Rβ/γ complex is a common intermediate affinity receptor for IL-2 and IL-15 (Kd=1 nM), and both cytokines compete to bind to this receptor (7). Cytokine specificity is conferred by additional private chains, IL-2Rα and IL-15Rα, that are structurally related (8). These two chains contain structural domains (called sushi domains) previously found in some complement and adhesion molecules (9). IL-2Rα contains two such domains, whereas IL-15Rα contains only one. One noticeable difference is that IL-2 binds to its specific IL-2Rα with an affinity (Kd=10 nM) far lower than IL-15 to IL-15Rα (Kd=0.05 nM). Each specific chain can associate with the IL-2Rβ/γ complex to form a cytokine-specific, functional high-affinity (αβγ) receptor (10-12).
Due to the sharing of this IL-2Rβ/γ complex, both cytokines trigger similar downstream signaling pathways including activation of Jak-1/Jak-3 tyrosine kinases and subsequent nuclear translocation of the phosphorylated Stat-3 and Stat-5, activation of Lck and Syk tyrosine kinases, activation of the MAP kinase pathway, and induction of Bcl-2 (13,14). In contrast to IL-2Rβ and IL-2Rγ that are required for signal transduction, the specific receptors IL-2Rα and IL-15Rα have short intracellular tails (13 and 41 amino-acids respectively) and IL-2Rα is considered to play no role in signal transduction. While initial studies have pointed out the dispensable role of the intracellular tail of IL-15Rα in signaling (8), more recent data suggest that IL-15Rα might mediate certain intracellular functions (15-17).
In contrast to the general functional redundancy observed in vitro, several findings point to complementary and even opposing actions of IL-2 and IL-15 in vivo. Indeed, whereas IL-2 and IL-2Rα gene expression is mainly restricted to the activated T cell compartment, IL-15 and IL-15Rα transcripts are expressed by various cell types and tissues (monocytes, dendritic and stromal cells, keratinocytes, placenta, skeletal muscle, heart) suggesting additional roles for the IL-15 system beyond the immune system (7,8). Cells expressing IL-15Rα in the absence of IL-2Rβ and/or IL-2Rγ have been described and some of them respond to IL-15 (17,18), suggesting the existence of new functional IL-15 receptor complexes not involving IL-2Rβ and/or IL-2Rγ.
Distinct roles for IL-2 and IL-15 are also suggested from experiments in knock-out mice. While IL-2−/− and IL-2Rα−/− mice develop exacerbated T and B cell expansion associated with autoimmune manifestations, IL-15−/− and IL-15Rα−/− mice on the contrary have normal T and B cell populations and display a profound defect in NK cells, NK-T cells, intraepithelial lymphocytes and CD8+ memory T cells (19,20). A recent study suggests that, contrary to the results obtained in vitro, the major role of IL-2 in vivo is to limit continuous expansion of activated T cells, whereas IL-15 is critical for initiating T cell division (21).
A number of studies have contributed to the identification of human disorders in which targeting the IL-15 system is of clinical relevance and potential benefit. Among them are autoimmune and inflammatory diseases, infectious diseases, transplant rejection, cancer and immunodeficiencies (22,23). In this context, the rational design of agonists or antagonists of the IL-15/receptor system is a major concern and requires a precise knowledge of the structure of the high-affinity IL-15 receptor complex.
A number of mutagenesis studies of human and murine IL-2 have led to the identification of several residues implicated in the binding to the IL-2Rα, β and γ chains. From these studies, residues K35, R38, F42 and K43, all located in the A-B loop of human IL-2, are involved in its binding to the IL-2Rα chain, whereas residues D20 on helix A and N88 on helix C are involved in the binding to the IL-2Rβ chain, and Q126 on helix D is crucial for binding to the IL-2Rγ chain (24-26). Similar regions were identified on mouse IL-2 (27).
On the contrary, very little data is available concerning the residues on IL-15 involved in the binding to the different IL-15 receptors.
Some mutations in human IL-15 (D8 and Q108) which are analogous to the ones described for human IL-2 suggested that the corresponding regions in IL-15 are involved in the binding to the IL-2Rβ and γ subunits, respectively (28).
The present invention follows different complementary approaches including ligand receptor interaction analysis, induction of biological activity, peptide scanning, and site-directed mutagenesis, to define the epitope of IL-15 responsible for high-affinity binding to the IL-15Rα chain.